• Usha K Srinivas

      Articles written in Journal of Biosciences

    • Variable association of DNA polymerase with the cytoskeletal fractions of resting and dividing cells

      Usha K Srinivas C S Shashikant M R Das

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      A DNA polymerase activity associated with the detergent insoluble cytoskeletal fraction has been identified in dividing and non-dividing rat hepatocytes and a hepatoma (the Zajdela Ascitic Hepatoma). About 35 % of the enzyme is found associated with the cytoskeletal fraction of non-dividing cells as compared to about 3–6 % of the enzyme in dividing cells even though the dividing cells contain larger amounts of the extranuclear enzyme. The properties of the enzyme are similar to those of DNA polymerase-v. It is suggested that the association of the enzyme with the cytoskeletal fraction has functional significance.

    • Changes in cytoplasmic pH upon heat shock in embryonic and adult rat liver ceils

      Usha K Srinivas C J Revathi

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      All living cells, when exposed to elevated temperatures, undergo physiological changes which result in the expression of a specific set of heat shock proteins. Study of the possible physiological changes in adult and embryonic rat liver cells indicated a change in intracellular pH upon heat shock. Using 2′, 7′-bis (2-carboxyethyl)-5 (and -6) carboxyfluorescein acetoxymethyl ester, we demonstrate here that the intracellular pH of adult and embryonic liver cells is different and that there is an increase in relative fluorescence intensity in both adult and embryonic cells upon heat shock, which corresponds to about 0.2 to 0.3 pH units. We also show that in addition to heat, some of the inducers of heat shock like response in many systems also induce a change in intracellular pH and induce heat shock proteins at 37°C in fetal liver cells. The possible mechanisms of induction of heat shock proteins during heat shock and in the presence of inducers at normal temperature are discussed.

    • Role of heat shock transcription factors in stress response and during development

      Usha K Srinivas S K Swamynathan

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      A variety of physical and chemical stimuli were known to cause specific stress response in all organisms. Research done over the past 25 years has shown that there is a striking uniformity in the manner in which organisms respond to different forms of stress. Expression of stress responsive genes was shown to be regulated by two or more specific transcription factors present in the cell prior to stress. These specific genes were also activated during development. In this review, the role of heat shock transcription factors in stress response, during development and during cell cycle is described.

    • Cloning and characterization of mouse cullin4B/E3 ubiquitin ligase

      Rachana Tripathi K Seetharama Sastry Satya Keerthi Kota Usha K Srinivas

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      Heat induced differentiation of mouse embryonal carcinoma cells PCC4 has been reported earlier. We have further characterized the phenotype of the differentiated cells and by DD-RT-PCR identified several partial cDNAs that are differentially expressed during differentiation. Nucleotide homology search revealed that the genes corresponding to some of the up-regulated partial cDNAs are indeed part of differentiation pathway. 5′ extension of an EST that has homology to one of the partial cDNAs led to the identification of mouse cullin4B. Cullin4B is coded by a separate gene and has a unique and longer amino-terminal end with a putative nuclear localization signal sequence (NLS). We have cloned, expressed and raised antibodies against the amino and carboxy-terminal halves of cullin4B. Immuno staining of differentiated PCC4 cells with N-terminal Cul4B antibody showed enhanced expression of Cul4B and its translocation into the nucleus upon differentiation. Transient transfection of a chimeric gene encoding the N-terminal part of Cul4B fused to green fluorescent protein into PCC4 cells revealed that the protein was localized in the nucleus confirming the functional significance of the putative NLS. Since cullins are involved in recognition of specific proteins for degradation, based on the evidence presented here, we hypothesize that cullin4B is probably involved in differentiation specific degradation/ modification of nuclear proteins.

    • Heat shock transcription factors regulate heat induced cell death in a rat histiocytoma

      Kolla V, P Rasad Aftab Taiyab D Jyothi Usha K Srinivas Amere S Sreedhar

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      Heat shock response is associated with the synthesis of heat shock proteins (Hsps) which is strictly regulated by different members of heat shock transcription factors (HSFs). We previously reported that a rat histiocytoma, BC-8 failed to synthesize Hsps when subjected to typical heat shock conditions (42°C, 60 min). The lack of Hsp synthesis in these cells was due to a failure in HSF1 DNA binding activity. In the present study we report that BC-8 tumor cells when subjected to heat shock at higher temperature (43°C, 60 min) or incubation for longer time at 42°C, exhibited necrosis characteristics; however, under mild heat shock (42°C, 30 min) conditions cells showed activation of autophagy. Mild heat shock treatment induced proteolysis of HSF1, and under similar conditions we observed an increase in HSF2 expression followed by its enhanced DNA binding activity. Inhibiting HSF1 proteolysis by reversible proteasome inhibition failed to inhibit heat shock induced autophagy. Compromising HSF2 expression but not HSF1 resulted in the inhibition of autophagy, suggesting HSF2 dependent activation of autophagy. We are reporting for the first time that HSF2 is heat inducible and functions in heat shock induced autophagic cell death in BC-8 tumor cells.

    • Cullin4B/E3-ubiquitin ligase negatively regulates 𝛽-catenin

      Rachana Tripathi Satya Keerthi Kota Usha K Srinivas

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      𝛽-catenin is the key transducer of Wingless-type MMTV integration site family member (Wnt) signalling, upregulation of which is the cause of cancer of the colon and other tissues. In the absence of Wnt signals, 𝛽-catenin is targeted to ubiquitin–proteasome-mediated degradation. Here we present the functional characterization of E3-ubiquitin ligase encoded by cul4B. RNAi-mediated knock-down of Cul4B in a mouse cell line C3H T10 (1/2) results in an increase in 𝛽-catenin levels. Loss-of-function mutation in Drosophila cul4 also shows increased 𝛽-catenin/Armadillo levels in developing embryos and displays a characteristic naked-cuticle phenotype. Immunoprecipitation experiments suggest that Cul4B and 𝛽-catenin are part of a signal complex in Drosophila, mouse and human. These preliminary results suggest a conserved role for Cul4B in the regulation of 𝛽-catenin levels.

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